Eukaryotic cells use microtubule-based transport to accomplish intracellular organelle movement, cell division, and possibly cellular morphogenesis. These events require the activities of microtubule-motor proteins such as kinesin. In this proposal we describe experiments designed to understand how kinesin converts chemical energy into mechanical force, how kinesin attaches to the elements that it moves, what structural features of kinesin are essential for in vivo function, and where kinesin fits in the scheme of all MT-based movements, some of which might be generated by kinesin-like motors. To accomplish these goals, we will: 1) continue our studies of the elements of the kinesin heavy chain motor domain needed for force generation and develop methods for large-scale production of the kinesin motor domain for use in high-resolution structural studies; 2) begin analyses of proteins with which the kinesin heavy chain tail interacts in order to attach to cellular cargoes. 3) alter kinesin heavy chain in defined ways and introduce these altered molecules back into the organism for study in vivo; and 4) analyze five new genes that we have recently discovered that appear to encode kinesin-like proteins. In toto, our investigations will reveal how one particular microtubule motor functions and will begin to unravel the range of functions and strategies used by eukaryotes to accomplish microtubule-based movements.